Ever since the discovery of insulin in 1922 many different types of insulin preparations have been used for the treatment of Diabetes mellitus. At the beginning exclusively insulin solutions exhibiting a rapidly commencing and relatively rapidly ceasing insulin activity were used, but later on insulin preparations exhibiting a wider profile of activity procured by lowering the solubility of insulin by means of additions as e.g. zinc salt and/or protamines have been produced. For reasons of availability the insulin used herefor has normally been recovered from Pancreas from domestic animals, most frequently oxes, pigs and sheep, however, recently preparations containing human insulin of biotechnological origin have also appeared on the market.
The structure of human insulin is shown in the following formula ##STR1##
The insulins from certain domestic animals are very similar in structure to human insulin. Thus dog and pig insulin differ from human insulin only by containing Ala in position 30 in the B-chain and rabbit insulin only by containing Ser in the same position. These insulins may be converted into human insulin by replacement of the B30-amino acid residue with Thr by semisynthetic procedures as described by Morihara et al, Nature 280 (1979), 412-413 and Marcussen (U.S. Pat. No. 4,343,898).
When such an insulin is dissolved at physiological pH value a concentration-dependent association equilibrium is established between monomeric, dimeric, tetrameric, hexameric and even polymeric insulin. The equilibrium can e.g. be determined by ultracentrifugation, by osmometry or by gel filtration methods, vide e.g. R. Valdes Jr. and G. A. Ackers, "Methods in enzymology", vol. 61 (Enzyme Structure, part H. eds.; Hirs & Timasheff), Academic Press 1979, pages 125-142. In normal formulations of insulin preparations this equilibration is shifted in such a way that the insulin to a very high degree is on a hexameric form.
Substitutions in the insulin molecule can be introduced with the purpose of improving the profile of activity of the insulin in the treatment of Diabetes. Thus, Published International Application No. WO 86/05497 discloses that one or more substitutions of Glu in the insulin molecule by a neutral amino acid residue causes a shifting of the zone of precipitation of the insulin in such a way that a slow release after injection is obtained.
Moreover, Published European Application No. EP 214 826 discloses insulin analogues being particularly rapidly absorbed after injection. This effect is a result of the fact that by means of certain substitutions in particular in the B9-B12 region and in the B26-B28 positions in the insulin molecule a suppression of the association tendency of the insulin is obtained so that it is essentially present as monomer or dimer. However, a number of these insulin analogues exhibits a reduced biological activity.
Throughout the years a large number of artificially prepared analogues of human insulin has been described, usually with the purpose of elucidating the influence of the structure on the activity, vide e.g. Marke et al., Hoppe-Seyler's Z. Physiol Chem. 360 (1979), 1619-1632. Investigations of the influence of substitutions in the (B22-B26)-sequence of the insulin on the receptor binding have been of particular interest, as said sequence is considered to be an essential site of binding for the insulin receptor, and as naturally occurring mutations have been found with substitutions in said site. Vide e.g. S. Shoelson et al. PNAS 80 (1983), 7390-7394 and M. Kobayashi et al.: Biomed. Res. 5 (3) (1984), 267-272. Very low biological activities were found for analogues in which Phe (B24) or Phe (B25) are substituted, and therefore it was concluded that the presence of these two amino acids is of decisive importance to the receptor binding
The present invention is based on the surprising recognition that certain human insulin analogues in which one of the amino acid residues [Phe.sup.B24 ] or [Phe.sup.B25 ] is not present exhibit a low association tendency in solution and at the same time exhibits an unchanged or even higher in vitro biological activity than human insulin. The deletion of either [Phe.sup.B24 ] or [Phe.sup.B25 ] will have the effect that [Lys.sup.B29 ] is transferred into [Lys.sup.B28 ]. The position of a positive charge in this position in the human insulin molecule is considered to be the important aspect of the present invention.